The delivery of genes of therapeutic benefit to target cells is central to the concept of gene therapy. If gene therapy is to become a routine procedure it is of utmost importance that systems are developed that allow effective in vivo delivery of therapeutic genes to the target cells.
Viral vectors, and especially retroviral vectors are the most commonly used delivery vehicles for gene therapy (Morgan, R. A. and Anderson, W. F., Ann. Rev. Biochem., 62:191-217, (1993)). Most of the currently approved gene therapy protocols take an ex vivo approach in that cells are removed from the patient, genetically modified in vitro and then reintroduced into the patient. This procedure is cumbersome, expensive and limited to technologically advanced facilities. Further this kind of approach is limited to cells that can easily be isolated, cultured and reimplanted (Gxc3xcnzburg, W. H., and Salmons, B., Biologicals, 23:5-12 or Gxc3xcnzburg, W. H., and Salmons, B., Molecular Medicine Today, 1:410-417 1995). Although an in vivo delivery of therapeutic genes would offer many advantages, in its present form this approach is both inefficient and problematic. A major problem, due to low efficiency of gene transfer, is the necessity for multiple applications of viral vector. The requirement for multiple rounds of vector delivery is not only tedious but also likely to be unsuccessful because of immune responses directed towards the virus particles.
One possible way in which these problems could be circumvented is through the direct implantation of cells producing viral particles. The implantation of cells producing viral particles containing the genome of a viral vector in situ next to the target organ or cells would also allow direct application of the viral vector to the target cells/organs.
Additionally, where the viral vector virus used is a retroviral virus, such an approach have an advantage over multiple single high dosage applications, since the chances of the vector virus being present at the time when a target cell undergoes replication, and thus being able to infect the target cell is increased. Furthermore a lower but continuous release of viral particles may be advantageous in escaping host immune response against the viral particle.
For an effective delivery of viral vectors, the cells producing viral particles should be able to survive long periods in the host after implantation, and viral particles must be produced during this period and released from the cells. In the absence of a significant immune response, for instance after implantation in the brain, these cells can survive over prolonged periods (Culver et al., Science, 256:1550-1552 (1992); Ram, Z. et al., Cancer Res., 53:83-88 (1993)). However to achieve successful implantation at other sites in the body, the producer cells must be protected from the immune system.
The long term effectivity of this approach thus depends on (1) protection of the cells from the host immune system, which will normally eliminate cells producing viral particles, especially if the cells producing the viral particles are from a different species as is usually the case for such cells and (2) survival of the cells in situ for extended periods, which may require vascularization.
Encapsulation of cells in permeable structures that allow the release of certain biologically active molecules but protects the cells producing these molecules from the host immune system has met with some success (for a review see Chang, P. L., In somatic Gene Therapy, P. L Chang, ed. (CRC Press, Boca Raton) p. 203-223 (1995)). Cells that have been genetically modified to produce human growth hormone (hGH) (Tai, I. T. and Sun, A. M. FASEB J., 7:1061-1069 (1993)) or a secreted form of human adenosine deaminase (Hughes, M. et al., Hum. Gene Ther., 5:1445-1455 (1994)) have been encapsulated. In both of these studies, cells were encapsulated in poly-L-lysine-alginate microcapsules and the cells were shown to survive for long periods in culture. This was accompanied by long term production of the enzyme or hormone. Further, it was shown (Tai, I. T. and Sun, A. M., FASEB J., 7:1061-1069 (1993)) that upon transplantation of the microcapsules into mice, the cells remained viable for 1 year and they continued to produce hGH, demonstrating that the capsules protect the transfected cells from destruction by the host immune system.
Cell encapsulation has also been reported using other materials. Baby hamster kidney cells genetically modified to produce nerve growth factor have been encapsulated in polyacrylonitrile/vinyl chloride and implanted in rat brain. The encapsulated cells survived for at least 6 months and continued to produce NGF (Winn, S. R. et al., Proc. Natl. Acad. Sci. USA, 91:2324-2328 (1994); Deglon, N. et al., Gene Ther., 2:563 (1995)).
Additionally, hepatocytes have successfully been encapsulated in a polyelectrolyte complex of cellulose sulphate and polydimethyldiallyl ammonium (Stange, J. et al., Biomat. Art. Cells and Immob. Biotech., 21:343-352 (1993)). More that 90% of the encapsulated hepatocytes retained their viability and in contrast to hepatocytes grown as monolayers, the encapsulated cells showed an increased metabolic activity. It is not suggested herein that cellulose sulphate/polydimethyldiallylammonium capsules could support the growth other types of cells, such as cells producing viral particles, or allow the exit of viral particles from such capsules.
The preparation of cellulose sulphate capsules used in the present invention has been thoroughly described in DE 40 21 050 A1. Also the synthesis of the cellulose sulphate has been described in this patent application. Methods for a comprehensive characterization of cellulose sulphate capsules have been extensively dealt with in H. Dautzenberg et al., Biomat., Art. Cells and Immob. Biotech., 21(3):399-405 (1993). Other cellulose sulphate capsules have been described in GB2 135 954. The properties of the cellulose capsules, i.e. the size, the pore size, wall thickness and mechanical properties depend upon several factors such as for example physical circumstances whereunder the capsules have been prepared, viscosity of precipitation bath, its ion strength, temperature, rapidity of addition of cell/cellulose sulphate suspension, constitution of cellulose sulphate, as well as other parameters described by the Dautzenberg group.
It has surprisingly been found that the continuous production of viral particles from implanted cells can be achieved by encapsulation of the cells in a polyelectrolyte complex. Although the pores of such capsules are large enough to allow antibodies and complement, known to inactivate virus (Welsh, R. M. et al., Nature, 257:612-614 (1975); Cornetta, K. et al., Hum. Gene Ther., 1:15-30 (1990)), to enter the capsules, we have found no evidence of gross immune or inflammatory responses, or of necrosis in the vicinity of implanted capsules. Additionally, it has surprisingly been found that the capsules according to the present invention become well engrafted in the host, and become rapidly vascularized. The encapsulated cells according to the invention thus permits long term delivery of viral vectors carrying therapeutic genes in vivo.
The invention then, inter alia, comprises the following alone or in combination:
Encapsulated cells producing viral particles comprising a core containing cells; and a porous capsule wall surrounding said core, said porous capsule wall being permeable to said viral particles;
encapsulated cells as above wherein said porous capsule wall consists of a polyelectrolyte complex formed from counter-charged polyelectrolytes;
encapsulated cells as above wherein said porous capsule wall consists of a polyelectrolyte complex formed from sulphate group-containing polysaccharides or polysaccharide derivatives or sulphonate group-containing synthetic polymers and polymers with quaternary ammonium groups;
encapsulated cells as above wherein the sulphate group-containing polysaccharides or polysaccharide derivative is cellulose sulphate, cellulose acetate sulphate, carboxymethycellulose sulphate, dextran sulphate or starch sulphate, and the sulphonate group-containing synthetic polymer is a polystyrene sulphonate;
encapsulated cells as above wherein the polymer with quaternary ammonium groups is polydimethyldiallylammonium or polyvinylbenzyl-trimethylammonium;
encapsulated cells as above wherein the porous capsule wall consists of a complex formed from cellulose sulphate and polydimethyldiallyl ammonium;
encapsulated cells as above having the form of microcapsules with a diameter between 0.01 and 5 mm, preferably between 0.1 and 1 mm;
encapsulated cells as any above wherein said capsule consist a spongy cellulose sulphate matrix forming the interior of the capsule wall, surrounded by a capsule surface containing pores; said spongy matrix being filled with cells;
encapsulated cells as above wherein the surface pore size of the porous capsule wall is between 80 and 150 nm, preferably between 100-120 nm;
encapsulated cells as above wherein the viral particle produced by the encapsulated cells is a retroviral particle containing the genome of a retroviral vector;
encapsulated cells as above wherein the encapsulated cells producing retroviral particles is a packaging cell line transfected with an expression vector, said expression vector carrying a retroviral vector construct capable of infecting and directing the expression in target cells of one or more coding sequences carried by said retroviral vector construct; said packaging cell line harboring at least one expression vector carrying genes coding for the proteins required for the retroviral vector construct to be packaged;
encapsulated cells as above wherein at least one of said coding sequences code for heterologous peptides selected from marker genes, therapeutic genes, antiviral genes, antitumor genes, and cytokine genes;
encapsulated cells as above wherein said marker gene is selected from the group consisting of marker genes which codes for proteins such as xcex2-galactosidase, neomycin, alcohol dehydrogenase, puromycin, hypoxanthine phosphoribosyl transferase (HPRT), hygromycin and secreted alkaline phosphatase, and said therapeutic gene is selected from genes which codes for proteins such as Herpes Simplex Virus thymidine kinase, cytosine deaminase, guanine phosphoribosyl transferase (gpt), cytochrome P 450 and cell cycle regulatory genes such as SDI, tumour suppressor genes which codes for proteins such as p53 or antiproliferation genes which codes for proteins such as melittin, cecropin or cytokines such as IL-2;
encapsulated cells as above wherein the packaging cell line is selected from psi-2, psi-crypt, psi-AM, GP+E-86, PA317, and GP+envAM-12;
encapsulated cells as above wherein the expression vector transfected into the packaging cell line is pBAG, pLXSN, p125LX, pLX2B1, or pc3/2B1 or derivatives thereof;
a process for the preparation of encapsulated cells as above comprising suspending the cells providing viral particles in an aqueous solution of a polyelectrolyte, whereafter the suspension in the form of preformed particles is introduced into a precipitation bath containing an aqueous solution of a counter-charged polyelectrolyte;
a process as above wherein the particle formation takes place by spraying;
a process as above wherein the cells are suspended in an aqueous solution of a sulphate group-containing polysaccharide or polysaccharide derivative, or a sulphonate group-containing synthetic polymer;
a process as above wherein the sulphate group-containing polysaccharide or polysaccharide derivative is selected from cellulose sulphate, cellulose acetate sulphate, carboxymethylcellulose sulphate, dextran sulphate or starch sulphate, and the sulphonate group-containing synthetic polymer is a polystyrene sulphonate;
a process as above wherein the precipitation bath contain an aqueous solution of a polymer with quaternary ammonium groups;
a process as above wherein the polymer with quaternary ammonium groups is polydimethyldiallylammonium or polyvinylbenzyl-trimethylammonium;
a process as above wherein the cells are suspended in an aqueous solution of sodium cellulose sulphate, and introduced into a precipitation bath containing an aqueous solution of polydimethyldiallylammonium chloride;
a method as above wherein the aqueous cellulose sulphate solution is composed of 0.5-50%, preferably 2-5% sodium cellulose sulphate and 2-10%, preferably 5% fetal calf serum in buffered saline;
a method as above wherein the aqueous solution in the precipitation bath is composed of 0.5-50% preferably 2-10%, or more preferred 3% polydimethyldiallylammonium chloride in buffered saline;
encapsulated cells as any above produced by a process as above;
the use of the encapsulated cells as any above for the delivery of genes to target organ/cells comprising:
a) Culturing the encapsulated cells in a suitable medium, and
b) Implantation of the encapsulated cells into a living animal body, including a human;
the use as above wherein the target organ/cells is the mammary gland, or the pancreas; and
the use as above wherein the target organ/cells are the smooth muscle cells and other cell types surrounding the arteries.